The present invention relates to substrate analogs of a UDP-GlcNAc:muramyl pentapeptide pyrophosphoryl, N-acetylglucosaminyltransferase (GlcNAc transferase, MurG, or its homologs), an enzyme involved in bacterial cell wall biosynthesis. The substrate analogs of the invention are usefull as functional substitutes of Lipid I, the membrane bound, natural substrate of MurG. In particular, the substrate analogs of the present invention can be used advantageously in an assay for the enzymatic activity catalyzed by MurG, in methods for identifying other substrate analogs of MurG, as well as inhibitors of enzymatic activity or cell wall biosynthesis (i.e., potential antibacterial drugs), and in the isolation/purification of MurG, including studies of its active protein/peptide fragments.
2.1. Bacterial Enzymology
The emergence of resistance to existing antibiotics has rejuvenated interest in bacterial enzymology. It is hoped that detailed mechanistic and structural information about bacterial enzymes involved in critical biosynthetic pathways could lead to the development of new antibacterial agents. Because interference with peptidoglycan biosynthesis is a proven strategy for treating bacterial infections, all of the enzymes involved in peptidoglycan biosynthesis are potential targets for the development of new antibiotics. While some detailed structural and mechanistic information on some of the early enzymes in the pathway is now available, most of the downstream enzymes have proven very difficult to study.
There are two main reasons for this difficulty: First, the downstream enzymes are membrane-associated, making them intrinsically hard to handle; secondly, discrete substrates for most of the downstream enzymes are either not available or not readily so. In some cases monomeric substrates are difficult to obtain in large quantities from natural sources. In other cases substrates, which may be available in large quantities from natural sources, are intractable polymeric substances. In the absence of readily available discrete substrates, it has been impossible to develop enzyme assays that can be used to measure the activity of the downstream enzymes reliably and under a well-defined set of reaction conditions. This unfulfilled need has thwarted attempts to purify many of the downstream enzymes in an active form suitable for structural characterization, much less permitted attempts to obtain detailed mechanistic information on such enzymes.
Some of the best antibiotics function by interfering with the biosynthesis of the peptidoglycan polymer that surrounds bacterial cells. With the emergence of bacterial pathogens that are resistant to common antibiotics it has become imperative to learn more about the enzymes involved in peptidoglycan biosynthesis. Although remarkable progress has been made in characterizing some of the early enzymes in the biosynthetic pathway (See, e.g., (a) Fan, C.; Moews, P. C.; Walsh, C. T.; Knox, J. R. Science 1994, 266, 439; (b) Benson, T. E.; Filman, D. J.; Walsh, C. T.; Hogle, J. M. Nat. Struct. Biol. 1995, 2, 644; (c) Jin, H. Y.; Emanuele, J. J.; Fairman, R.; Robertson, J. G.; Hail, M. E.; Ho, H. T.; Falk, P.; Villafranca, J. J. Biochemistry 1996, 35, 1423; (d) Skarzynski, T.; Mistry, A.; Wonacott, A.; Hutchinson, S. E.; Kelly, V. A.; Duncan, K. Structure 1996, 4, 1465; (e) Schonbrunn, E.; Sack, S.; Eschenburg, S.; Perakis, A.; Krekel, F.; Amrhein, N.; Mandelkow, E. Structure 1996, 4, 1065. (f) Benson, T. E.; Walsh, C. T.; Hogle, J. M. Biochemistry 1997, 36, 806.), the downstream enzymes have proven exceedingly difficult to study. Part of the difficulty stems from the fact that such downstream enzymes are membrane-associated (See, e.g., (a) Gittins, J. R.; Phoenix, D. A.; Pratt, J. M. FEMS Microbiol. Rev. 1994, 13, 1; (b) Bupp, K.; van Heijenoort, J. 1993, 175, 1841.), making them intrinsically hard to handle, and partly because substrates for many of the enzymes are not readily available. (See, e.g., (a) Pless, D. D.; Neuhaus, F. C. J. Biol. Chem. 1973, 248, 1568; (b) van Heijenoort, Y.; Gomez, M.; Derrien, M.; Ayala, J.; van Heijenoort, J. J. Bacteriol. 1992, 174, 3549.) These problems have impeded the development of activity assays suitable for detailed mechanistic investigations of the downstream enzymes. For a fluorescent assay to monitor MraY activity, see: Brandish, P. E.; Burnham, M. K.; Lonsdale, J. T.; Southgate, R.; Inukai, M.; Bugg, T. D. H. J. Biol. Chem. 1996, 271, 7609.
2.2. MurG
One such downstream enzyme is MurG, which is involved in peptidoglycan biosynthesis. MurG catalyzes the last intracellular step in the biosynthetic pathway of peptidoglycan biosynthesis, i.e., the transfer of UDP-N-acetylglucosamine (UDP-GlcNAc) to the lipid-linked N-acetylmuramylpentapeptide substrate, Lipid I. (See, Scheme 1, below.)
Although the murG gene is first identified in E. coli in 1980 and is sequenced independently by two groups in the early 1990""s, very little is known about the MurG enzyme. There are no mammalian homologs, and no direct assays for MurG activity have been developed, in part because the lipid-linked substrate (Lipid I, Scheme 1) is extremely difficult to isolate. This lipid-linked substrate is present only in minute quantities in bacterial cells. Although it is possible to increase the quantities of lipid-linked substrate by using bacterial cells engineered to overexpress enzymes involved in the synthesis of the lipid-linked substrate, isolation remains very difficult. Moreover, the isolated substrate is hard to handle.
Consequently, MurG activity is currently assessed using crude membrane preparations by monitoring the incorporation of radiolabel from radiolabeled UDP-GlcNAc donor group into lipid-linked acceptor components in the membrane. To increase the signal, the membranes are often prepared from bacterial cultures that overexpress MraY and/or MurG. MraY is the enzyme that catalyzes the reaction that attaches the MraY substrate, UDP-N-acetyl muramic acid pentapeptide, to a lipid phosphate moiety to provide Lipid I, which is the substrate for MurG. Typically, the membrane preparations are supplemented with exogenous UDP-N-acetyl muramic acid pentapeptide for conversion to Lipid I. This MraY substrate can be readily isolated in large quantities from bacterial cultures. Although this xe2x80x9ccoupledxe2x80x9d enzyme assay is manageable for screening of potential inhibitors of the MurG enzyme, it is not suitable for detailed mechanistic investigations, and it cannot be used to follow MurG activity during purification.
More specifically, MurG is a cytoplasmic membrane-associated enzyme which catalyzes the transfer of UDP-N-acetylglucosamine (UDP-GlcNAc) to the C4 hydroxyl of an undecaprenyl pyrophosphate N-acetylmuramyl pentapeptide substrate (Lipid I), resulting in the assembly of the disaccharide-pentapeptide building block (Lipid II, Scheme 1), which is incorporated into polymeric peptidoglycan. See, e.g., (a) Bugg, T. D. H.; Walsh, C. T. Nat. Prod. Rep. 1992, 199; (b) Mengin-Lecreulx, D.; Flouret, B.; van Heijenoort, J. J. Bacteriol. 1982, 151, 1109. As already mentioned, the muramyl pentapeptide substrate is unique to bacteria Hence, the MurG enzyme is a potential target for the discovery or design of specific MurG inhibitors.
Despite decades of effort spent characterizing MurG activity, there is virtually no structural or mechanistic information on the enzyme. See, e.g., (a) Anderson, J. S.; Matsuhashi, M.; Haskin, M. A.; Strominger, J. L. Proc. Natl. Acad. Sci. USA 1965, 53, 881; (b) Anderson, J. S.; Matsuhashi, M.; Haskin, M. A.; Strominger, J. L. J. Biol. Chem. 1967, 242, 180; (c) Taku, A.; Fan, D. P. J. Biol. Chem. 1976, 251, 6154; (d) Mengin-Lecreulx, D.; Texier, L.; van Heijenoort, J. Nucl. Acid. Res. 1990, 18, 2810; (e) Ikeda, M.; Wachi, M.; Jung, H. K.; Ishino, F.; Matsuhashi, M. Nucl. Acid Res. 1990, 18, 4014; (f) Mengin-Lecreulx, D.; Texier, L.; Rousseau, M.; van Heijenoort, J. J. Bacteriol1991, 173, 4652; (g) Miyao, A.; Yoshimura, A.; Sato, T.; Yamamoto, T.; Theeragool, T.; Kobayashi, Y. Gene, 1992, 118, 147; (h) Ikeda, M.; Wachi, M.; Matshuhashi, M. J. Gen. Appl. Microbiol., 1992, 38, 53. Difficulties isolating Lipid I have prevented the development of a simple, direct assay for MurG activity. Consequently, it has not been possible to purify MurG in a quantifiably active form or to determine the minimal functional length; nor has it been possible to carry out any detailed mechanistic studies, or to determine the substrate requirements.
Therefore, there exists a need for a direct enzyme assay that can be used both for effective screening of enzyme inhibitors and for the purification, characterization and identification of MurG, its various mutants and active fragments thereof. 
Substrate analogs for MurG enzyme, a GlcNAc transferase, are disclosed. For the first time, a substrate analog of Lipid I, as shown above in Scheme 1, (i) having a structure that is accepted by at least wild type MurG enzyme such that a labeled coupling product is produced by the GlcNAc transferase activity of the enzyme in the presence of the substrate analog and labeled UDP-GlcNAc, and (ii) having structural features that facilitate the separation of labeled UDP-GlcNAc from the labeled coupling product.
In particular, a substance is described herein, which comprises the chemical moiety of the formula: 
in which xe2x80x9cRxe2x80x9d is an acyl group comprising 2 or more carbon atoms, xe2x80x9cR1xe2x80x9d is a substituted or unsubstituted alkyl group comprising 1 or more carbon atoms, xe2x80x9cR2xe2x80x9d is a hydrogen or a substituted or unsubstituted alkyl group comprising 1 or more carbon atoms, xe2x80x9cAxe2x80x9d is a substituted or unsubstituted amino acid residue or a peptide comprising 2 or more substituted or unsubstituted amino acid residues, xe2x80x9cR3xe2x80x9d is a substituted or unsubstituted alkyl group comprising 5 or more carbon atoms, the substance exhibiting a binding affinity for at least wild type MurG enzyme and provided that the substance is not Lipid I, the natural substrate of wild type MurG enzyme. More particularly, the substance of the invention serves as an acceptor for the GlcNAc transferase activity of at least wild type MurG enzyme or its homologs.
Also disclosed is a method of detecting GlcNAc transferase activity in a sample suspected of containing a protein or an active fragment thereof exhibiting GlcNAc transferase activity. Preferably the method comprises (a) providing a sample suspected of containing a protein or an active fragment thereof exhibiting GlcNAc transferase activity; (b) contacting the sample with effective amounts of labeled GlcNAc substrate and a substance comprising the chemical moiety of the formula (I), above, under conditions effective to provide a labeled coupling product comprising labeled GlcNAc coupled to the substance via a glycosidic bond in the presence of a protein or an active fragment thereof exhibiting GlcNAc transferase activity; and (c) detecting the formation or presence of the labeled coupling product, which is indicative of GlcNAc transferase activity in the sample.
It is also an objective of the present invention to provide an assay for detecting GlcNAc transferase activity in a sample suspected of containing a protein or an active fragment thereof exhibiting GlcNAc transferase activity comprising a compound of the formula (I), above. A screen and methods of utilizing same are also contemplated by the present invention. In particular, a screen is provided for compounds exhibiting potential antibacterial activity comprising (i) a protein or an active fragment thereof exhibiting GlcNAc transferase activity, (ii) a substance comprising the chemical moiety of the formula (I), above, and (iii) a labeled GlcNAc substrate.
Additionally, the method of this invention provides a detection step comprising binding the xe2x80x9cAxe2x80x9d or xe2x80x9cR3xe2x80x9d groups of formula 1 to a solid support via a biotin tag, wherein said solid support includes an avidin or streptavidin coated resin. This step provides a continuous monitoring of product formation via the use of scintillation proximity assay. Furthermore, the separation of biotin-labeled substance involves filtration through an avidin-coated resin.
In a preferred embodiment of the invention xe2x80x9cR3xe2x80x9d may be selected from H, an aliphatic group comprising 1 to about 50 carbon atoms, an aromatic or heteroaromatic group comprising 3 to about 55 carbon atoms, pyrophosphate protecting groups and pharmaceutically acceptable salts thereof.
Additionally, a method detection step comprises binding said xe2x80x9cAxe2x80x9d or xe2x80x9cR3xe2x80x9d to a solid support via a biotin tag, wherein said solid support includes an avidin or streptavidin coated resin.
Hence, substrate analogs are prepared, which are used in an enzyme assay for MurG or MurG-like activity. A direct assay for MurG activity is thus provided.
These and other objects of the invention are described further, below, along with the preferred embodiments of the invention.